A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

Shorter J, Warren G - J. Cell Biol. (1999)

Bottom Line:
Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process.Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking.These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

ABSTRACTDuring telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

Figure 6: Effect of antibodies against giantin and GM130 on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were either fixed and processed for EM, held on ice for 15 min, or preincubated on ice for 15 min with 1 μl of anti-GM130 NN15 serum, antigiantin serum, or a combination of both. The pretreated MGF were then incubated for 60 min at 37°C with: p97 (70 ng/μl); p47 (37.5 ng/μl) and p115 (30 ng/μl); or NSF (100 ng/μl), α-SNAP (25 ng/μl), γ-SNAP (25 ng/μl) and p115 (30 ng/μl); or all these components combined. Samples were fixed and processed for EM, and the percentage cisternal regrowth ± SEM and the percentage total membrane present as stacked regions of cisternae ± SEM was determined.

Mentions:
When the MGF were resuspended for the p97 or NSF/p97 combined pathway, cisternal regrowth was unaffected by antigiantin and/or anti-GM130 (Fig. 6 A). In contrast to this, the stacking process was severely inhibited in these two pathways (Fig. 6 B) and the preimmune sera had no effect on this process (data not shown). Thus, p115 stacking function requires p115 interactions with GM130 and giantin. Furthermore, this indicates that p115 may be able to tether cisterna to cisterna, as well as COPI vesicle to cisterna.

Figure 6: Effect of antibodies against giantin and GM130 on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were either fixed and processed for EM, held on ice for 15 min, or preincubated on ice for 15 min with 1 μl of anti-GM130 NN15 serum, antigiantin serum, or a combination of both. The pretreated MGF were then incubated for 60 min at 37°C with: p97 (70 ng/μl); p47 (37.5 ng/μl) and p115 (30 ng/μl); or NSF (100 ng/μl), α-SNAP (25 ng/μl), γ-SNAP (25 ng/μl) and p115 (30 ng/μl); or all these components combined. Samples were fixed and processed for EM, and the percentage cisternal regrowth ± SEM and the percentage total membrane present as stacked regions of cisternae ± SEM was determined.

Mentions:
When the MGF were resuspended for the p97 or NSF/p97 combined pathway, cisternal regrowth was unaffected by antigiantin and/or anti-GM130 (Fig. 6 A). In contrast to this, the stacking process was severely inhibited in these two pathways (Fig. 6 B) and the preimmune sera had no effect on this process (data not shown). Thus, p115 stacking function requires p115 interactions with GM130 and giantin. Furthermore, this indicates that p115 may be able to tether cisterna to cisterna, as well as COPI vesicle to cisterna.

Bottom Line:
Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process.Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking.These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

ABSTRACTDuring telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.